It is known that, in order to ascertain a possible pathology, diagnostic tests are used, and according to the results of these, the most appropriate therapy is begun.
In a clinical laboratory, at least three different pre-analytical protocols are normally provided, which use three different blood samples, collected in three different containers, so that at least three different groups of clinical tests can be carried out.
A first group of tests comprises physical tests to measure the erythrocyte sedimentation rate in the blood (ESR), for example using the method and apparatus described in the European patent application EP-A-1.098.188 (EP'188) in the name of the present Applicant, and to effect measurements of size of the corpuscular part of the blood, and also to determine values of anemia and hematocrit and/or hemoglobin, as described in the Italian patent application UD2006000111 in the name of the present Applicant.
The test to measure the ESR is indicative, non-specifically, of the presence of an inflammatory state, since it is known that, in particular pathological states, the red corpuscles tend to aggregate and form agglomerations, called rouleaux. This is typically shown by the ESR clinical test which has the advantage of being easy to carry out and inexpensive.
Aggregation is normally impeded by the negative charge of the red corpuscles, as a result of which the latter repel each other. However, it is possible that the negative charge may be neutralized when there are proteins with a positive charge present in the plasma, which therefore promote aggregation.
This explains the increase in the ESR in physiological or pathological situations which imply an increase in fibrinogen, beta globulins, alpha globulins and gamma globulins.
In collagen pathologies, rheumatic diseases and Hodgkin's disease, there is a connection between the ESR values and the level of activity of the disease: the ESR exam, in these cases, is a useful instrument for monitoring a patient's clinical conditions.
From the above, it is clear that the ESR indirectly reveals a state of phlogosis.
It is also clear that the non-specific nature of the ESR test makes it necessary to use it in the context of clinical and anamnestic data, which retain a primary role.
Therefore, when the ESR value exceeds normal values, it is consolidated practice to carry out other diagnostic tests, even if they are more expensive, but which are more specific.
Among these, a second group of tests, of the immunological type, that is, based on an antigen-antibody reaction, serves to assess the concentration of C-reactive protein (CRP), streptococcus infections (ASO) and the rheumatic factor RF.
The C-reactive protein CRP is a protein, present in the blood serum, which significantly increases following damage to the tissues, bacterial and viral infections, cardiac pathologies and malign neoplasia.
Recent years have seen important developments in the search for inflammatory markers which could act as predictors of the risk of cardiovascular events; the most credited hypothesis is that atherosclerosis is the result of an inflammatory process, which develops in response to metabolic damage (diabetes, hypercholesterolemia), physical damage (hypertension) or behavioral damage (smoking).
The clinical implications of measuring these markers consist in the possibility of evaluating the individual risk of cardiovascular events. The estimation of traditional predisposing factors (advanced age, diabetes, smoking, hypertension, hyperlipemia and previous angina) defines the risk on a population level quite accurately, but allows to predict only 50-60% of the variation in the absolute risk in the individual patient.
The importance has thus emerged of ascertaining the values of C-reactive protein as predictors of independent risk.
Known methods to determine CRP use the fact that, in a solution, an antibody recognizes and binds to a specific antigen, determining an immunological reaction that is detected in particular by observing the change in optical properties of the solution.
To exploit this effect and effectively investigate it by measuring the change in optical properties, the formation of a suspension or a precipitate has to be avoided, the formation of an aggregation or an agglutination being, vice versa, desirable.
In particular, in suitable conditions an immunological reaction can occur between specific antibodies immobilized on a suitable carrier, for example gold colloid or latex particles, and the antigens, the result of which is an aggregation or agglutination mixture that can be determined in its change in absorbance or other optical properties.
The standard test to determine CRP consists in measuring the level of turbidity caused by the agglutination due to the mixing of a determinate quantity of blood serum or plasma with latexes consisting of balls, mainly polystyrene, with an average diameter of about 0.120 micron, covered with an anti-CRP antibody and dispersed or diluted in particular liquids, called buffers.
The CRP test used in the state of the art is based on nephelometry and requires average times of about 6 minutes to achieve significant kinetics, since the nephelometric technique requires low-concentration reagents.
Other methods are disclosed, for example in the documents WO-A-89/06801 and WO-A-92/11537, that are based on gold colloid that forms superaggregated complexes, that may replace the latex particles used in immunoagglutination assay, detectable by a densitometer or a reflectometer.
Further methods use whole blood, instead of blood serum or plasma, and provide an initial lysis of the red corpuscles present and then agglutination due to the antigen present in the volume of the plasma, which meets the CRP-specific antibody present in the sensitized carrier. In this case, the value of the CRP measured is corrected with the hematocrit value of the whole blood sample.
A known method is described, for example, in the document “Rapid Immunometric Measurement of C-Reactive Protein in Whole Blood”, Petter Urdal, Stig M. Borch, Sverre Landaas, May B. Krutnes, Geir O. Gogstad, and Per Hjortdahl, published in Clinical Chemistry 38/4, 580-584 (1992).
In particular, this latter is related to the aforementioned documents WO-A-89/06801 and WO-A-92/11537 and it discloses an immunoassay method, commercially available as a kit called “NycoCard CRP Whole Blood test”, to determine the content of C-reactive protein (CRP) in a sample of whole blood that involves an immobilization of a CRP specific antibody onto a ultrasmall gold colloid that acts as a suitable carrier, a lysis treatment on the sample of whole blood, an immunological reaction between antigen and immobilized antibody and an investigation of the resulting aggregation or agglutination deriving from the gold colloid carrying the antibodies bound to the antigens, using a reflectometer.
From the European patent application EP-A-0.822.412 (EP'412) an immunoassay method is also known for detecting the content of C-reactive protein (CRP) in a whole blood sample that, as well as in the aforementioned document “Rapid Immunometric Measurement of C-Reactive Protein in Whole Blood”, involves an agglutination reaction in the sample between antigen and antibody immobilized on a polystyrene latex and an optical measurement of the change of absorbance of the agglutination mixture, wherein the whole blood sample is forcibly lysed.
In the course of infections produced by Streptococcus pyogenes, numerous substances are secreted, including two hemolysins: Streptolysin “O” and Streptolysin “S”. Streptolysin “O” is a toxin able to stimulate the production of specific antibodies. With the same principle of the CRP test immunological reactions are achieved for the quantitative determination of the anti_Streptolysin “O” antibodies, ASO tests.
The discovery of these antibodies has been very useful in order to diagnose streptococcal infections and the relative consequences associated with them, such as rheumatic fever and acute glomerulonephritis. The test is based on the reaction between the antibody and the particles of latex linked to Streptolysin “O”. The ASO values are determined kinetically according to a calibration curve made using a sample with a known ASO concentration according to a succession of scalar concentrations, using the techniques already known in the CRP test.
Furthermore, a test is made in order to quantify the rheumatic factor RF, which is a macroglobulin which agglutinates particles of latex sensitized with human gamma globulins, and can be found in most patients affected with evolutive polyarthritis. The latex agglutination test allows to differentiate this illness from articular rheumatism or rheumatic fever, where the rheumatoid factor is not present.
A third group of tests concerns coagulation and, in particular, the evaluation of the concentration of fibrinogen present in the blood. Coagulation depends on factors found in the plasma and the platelets, thrombin, prothrombin, thromboplastin.
The level of fibrinogen in the blood can vary in pathological situations, for example increasing in inflammations, rheumatisms, inflammations of the connective tissues, in the lymphoma (more than 5 g/l), during pregnancy, in some cases of nephrosis and in the event of burns. On the contrary it decreases if too much of it is consumed (hyperfibrinogenolysis), in cases of kidney failure and disseminated intravascular coagulation due to various shocks (septic shock, prostate cancer).
For example, the International patent application WO-A-90/08949 (WO'949) discloses a plurality of optical based methods to determine the coagulation properties of a blood sample.
It is also known, as reported in a study published in Clinical Chemistry, “Mistakes in a stat laboratory: types and frequency”, that the analytical flow of each clinical test has three main working steps, that is, a pre-analytical step, an analytical step and a post-analytical step.
According to this study, 68.2% of mistakes occur during the pre-analytical step, 13.3% during the analytical step and the remaining 18.5% during the post-analytical step.
It is also clear that the first and fundamental requirement that the clinic and the patient ask from those performing the analysis is the reliability of the analytical result.
From the International patent application WO-A-2005/022125 (WO'125) in the name of the present Applicant an integrated apparatus is also known that comprises, arranged in line and integrated in a single machine, a device of the optical type to detect the speed of blood sedimentation (ESR) of a blood sample and a measuring assembly with a cell-counter function. The apparatus comprises one or more containers suitable to be perforated by a pick-up needle, in order to pick up the sample to be analyzed, in a quantity of between 30 μl and 200 μl. The sample is sent, by means of a pump and trough a circuit, to the analytical devices.
Such an apparatus is directed to the analysis of simple physical properties of the blood sample and all the components are developed keeping this aim in mind, for example, it allows only a rough control of the dosage of the sample and of the quantity of the sample that is sent to the analytical devices, nor does it make a pre-treatment on the whole blood sample to prepare the same for the subsequent analysis.
One purpose of the present invention is to achieve an apparatus that allows to carry out a plurality of diagnostic tests, which use different reaction techniques and a single sample of whole blood which has been subjected to a single pre-analytical treatment, in order to achieve the various reaction phenomena to be studied, both physical, immunological and coagulative.
Another purpose is to perfect a method that intrinsically reduces the probabilities of error in the pre-analytical step, which is economical, allowing to perform a plurality of diagnostic tests which use different reaction techniques and a single sample of whole blood which has been subjected to a single pre-analytical treatment, in order to achieve the various reaction phenomena to be studied, both physical, immunological and coagulative.
The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.